The A118 (Figure 13.2.A) and PSA are temperate phages and belong to the Siphoviridae family, characterized by dsDNA-containing isometric capsids and long, flexible tails.
The A118 genome consists of 40,834 bp encoding 72 open reading frames (ORFs). The DNA packaged into phage heads is larger and consists of approximately 43.3 kb, which indicated about 6% redundancy (Loessner et al. 2000). Circular permutation along with terminal redundancy is common among phages and requires the circularization of the phage genome after entry into the host cell. Evidence indicated that after rolling circle replication of A118 phage DNA, sequential packaging starts at a random point of the concatamer, and genomes exceeding the one unit genome size by approximately 2.5 kb of DNA are incorporated into the heads.
Based on sequence similarities, functions could be assigned to 26 of the A118 ORFs. Clustering of genes reflecting different life cycles is common in phages, and especially pronounced in temperate phages. These gene clusters can be divided into those involved in DNA replication, phage morphogenesis, and lysis and those genes necessary for the establishment and maintenance of lysogeny which (as in A118 and PSA) are often oriented in the opposite direction of transcription. Many temperate phages with the same hosts have significant homology over parts of their genomes. Superinfection of lysogenized hosts is common and results in a close encounter of different virus genomes. The theory introducing modular evolution of phage genomes has been postulated more than 25 years ago (Susskind and Botstein 1978) and has since been supported by detailed study of many phages. However, except for a similar arrangement of gene clusters, surprisingly little overall similarity exists between the genomes of A118 (serovar 1/2 host strains) and PSA (Zimmer et al. 2003), the latter of which only infects L. monocytogenes serovar 4 strains. This suggests that these phage lineages may have diverged at an earlier stage. Consequently, because phages can only evolve together with their host bacteria, one must assume that the corresponding host strain lineages also diverged relatively early.
While A118 integrates into a region homologous to the Bacillus subtilis comK gene which is then disrupted, the integration site of PSA is a t-RNAArg gene, where the attB is functionally complemented by prophage nucleotides. These sites appear to be frequently used by different Listeria phages to integrate, and possible ramifications on host pathogenesis will be discussed at a later stage. t-RNA genes are generally known to harbor prophages, in many different bacteria (Campbell 2003).
The 37,618-bp PSA genome is not circularly permuted nor is it terminally redundant, but features 3'overhanging ends of 10bp. Such cohesive (cos) ends are the alternative to circular permutation and allow direct circularization of the genome after entry into the host, without the requirement for a recombinase. Of the 57 ORFs found in the PSA genome, functional assignments could be made for 33 putative and confirmed gene products based on sequence homologies to known genes. Gene products from all life cycle-specific regions were identified, and, although there is little homology, they appear to serve similar functions as in A118. Two genes differ radically between the two phages. Because of their different substrates, the phage integrases are completely unrelated. Whereas the A118 integrase is a serine recombinase similar to Tn10 resolvase and Salmonella Hin invertase, the PSA integrase is homologous to the E. coli XerD protein. XerD is involved in resolving multimeric plasmids containing a xer site (Alen et al. 1997; Loessner and Calendar 2006). The endolysins responsible for host cell-wall degradation in lysis encoded by the two phages also differ. Endolysins have a two-domain organization, dividing the enzyme into two functional parts. The C-terminal part is responsible for substrate recognition, and binding to the cell wall is serovar-specific. The cell-wall binding domain (CBD) of phage PSA recognizes ligands on cell walls of serovars 4, 5, and 6, and the A118 CBD binds to serovar 1/2 and 3 cell walls. Both CBDs lack known motifs involved in the recognition of cell-wall anchors. The N-terminal, enzymatically active domains (EAD) also differ significantly from each other. The PSA EAD is an N-acetylmuramoyl-L-alanine amidase (Zimmer et al. 2003; Korndoerfer et al. 2006), whereas the A118 EAD is L-alanine-D-glutamate endopeptidase (Loessner et al. 1995). Interestingly, the endolysin of A500, another serovar 4b-specific phage, has a CBD which is almost identical to that of PSA, but its EAD is a peptidase related to Ply118. This supports the theory of modular evolution, with genetic exchange between phages. Phage endolysins have been employed to design attenuated suicide Listeria monocytogenes strains for delivery of antigen-encoding eukaryotic expression vectors (Dietrich et al. 1998). Phage endolysins and their applications with a focus on Listeria phage endolysins have recently been reviewed (Loessner 2005).
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